System for cell-based screening

ABSTRACT

The invention relates to an optical system for determining the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for specific biological activity. The invention involves providing cells containing fluorescent reporter molecules in an array of locations and scanning numerous cells in each location with a fluorescent microscope, converting the optical information into digital data, and utilizing the digital data to determine the distribution, environment or activity of the fluorescently labeled reporter molecules in the cells. The array of locations may be an industry standard 96 well or 384 well microtiter plate or a microplate which is a microplate having a cells in a micropatterned array of locations. The invention includes apparatus and computerized method for processing, displaying and storing the data.

This application is a continuation of U.S. application Ser. No.10/411,635, filed Apr. 11, 2003, now U.S. Pat. No. 7,235,373 which is acontinuation of U.S. application Ser. No. 09/293,210, filed Apr. 16,1999, now U.S. Pat. No. 6,620,591, which is a continuation of U.S.application Ser. No. 08/810,983, filed Feb. 27, 1997, now U.S. Pat. No.5,989,835. This application is related to pending U.S. applications Ser.Nos. 10/686,161, filed Oct. 15, 2003; 10/685,737, filed Oct. 15, 2003;09/718,770, filed Nov. 22, 2000; 10/411,635, filed Apr. 11, 2003; and11/229,382, filed Sep. 16, 2005.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of introducing fluorescent reagents intoor applying fluorescent reagents to cells and monitoring thefluorescence in the cells.

2. Description of the Prior Art

Drug discovery is a long, multiple step process involving theidentification of specific disease targets, development of an assaybased on a specific target, validation of the assay, optimization andautomation of the assay to produce a screen, high throughput screeningof compound libraries using the assay, hit validation and hit compoundoptimization. The output of this process is a lead compound that goesinto preclinical and eventually clinical trials. In this process, thescreening phase is distinct from the assay development phases andtesting the efficacy of the compounds in living biological systems.

Performing a screen on many thousands of compounds requires parallelhandling and processing of many compounds and assay component reagents.Standard high throughput screens use homogeneous mixtures of compoundsand biological reagents along with some indicator compound loaded intoarrays of wells in standard microtiter plates with 96 or 384 wells. Thesignal measured from each well, either fluorescence emission, opticaldensity, or radioactivity, integrates the signal from all the materialin the well giving an overall population average of all the molecules inthe well. This type of assay is commonly referred to as a homogeneousassay.

Science Applications International Corporation (SAIC) 130 Fifth Avenue,Seattle, Wash. 98109 describes an imaging plate reader. This system usesa CCD camera to image the whole area of a 96 well plate. The image isanalyzed to calculate the total fluorescence per well for homogeneousassays.

Molecular Devices, Inc. describes a system (FLIPR) which uses low anglelaser scanning illumination and a mask to selectively excitefluorescence within approximately 200 microns of the bottoms of thewells in standard 96 well plates in order to reduce background whenimaging cell monolayers. This system uses a CCD camera to image thewhole area of the plate bottom. Although this system measures signalsoriginating from a cell monolayer at the bottom of the well, the signalmeasured is averaged over the area of the well and is therefore stillconsidered a homogeneous measurement, since it is an average response ofa population of cells. The image is analyzed to calculate the totalfluorescence per well for cell-based homogeneous assays.

Proffitt et. al. Cytometry 24: 204-213 (1996) describes a semiautomatedfluorescence digital imaging system for quantifying relative cellnumbers in situ in a variety of tissue culture plate formats, especially96-well microtiter plates. The system consists of an epifluorescenceinverted microscope with a motorized stage, video camera, imageintensifier, and a microcomputer with a PC-Vision digitizer. TurboPascal software controls the stage and scans the plate taking multipleimages per well. The software calculates total fluorescence per well,provides for daily calibration, and configures easily for a variety oftissue culture plate formats. Thresholding of digital images andreagents which only fluoresce when taken up by living cells are used toreduce background fluorescence without removing excess fluorescentreagent. For example, in this system cells are pretreated withfluorescein diacetate (FDA) and loaded into 96-well plates.

Fluorescence microscopy of cells and tissues is well known in the art. Avariety of methods have been developed to image fluorescent cells in amicroscope and extract information about the spatial distribution andtemporal changes occurring in these cells. An article by Taylor, et al.in American Scientist 80 (1992), p. 322-335 describes many of thesemethods and their applications. These methods have been designed andoptimized for the preparation of a few specimens for high spatial andtemporal resolution imaging measurements of distribution, amount andbiochemical environment of the fluorescent reporter molecules in thecells.

Treating cells with dyes and fluorescent reagents and imaging the cellsis well known in the art. There is also a considerable body of prior artrelated to genetic engineering of cells to produce fluorescent proteins,such as modified green fluorescent protein (GFP) as a reporter molecule.The green fluorescent protein (GFP) of the jellyfish Aequorea victoriais a protein with an excitation maximum at 395 nm and an emissionmaximum at 510 nm and does not require an exogenous factor. Uses of GFPfor the study of gene expression and protein localization are discussedin more detail in papers by Chalfie et al. in Science 263 (1994), p.12501-12504. Some properties of wild-type GFP are disclosed by Morise etal. in Biochemistry 13 (1974), p. 2656-2662, and Ward et al. inPhotochem. Photobiol. 31 (1980), p. 611-615. An article by Rizzuto etal. in Curr. Biology 5 (1995), p. 635-642 discusses the use of wild-typeGFP as a tool for visualizing subcellular organelles in cells. A paperby Kaether and Gerdes in Febs Letters 369 (1995), p. 267-271, reportsthe visualization of protein transport along the secretory pathway usingwild-type GFP. The expression of GFP in plant cells is discussed by Huand Cheng in Febs Letters 369 (1995), p. 331-334, while GFP expressionin Drosophila embryos is described by Davis et al. in Dev. Biology 170(1995), p. 726-729. U.S. Pat. No. 5,491,084 describes expressing GFPfrom Aequorea victoria in cells as a reporter molecule fused to anotherprotein of interest. PCT/DK 96/00052 relates to methods of detectingbiologically active substances affecting intracellular processes byutilizing a GFP construct having a protein kinase activation site.Numerous references are related to GFP proteins in biological systems.For example, PCT/US94/10165 describes a system for isolating cells ofinterest utilizing the expression of a GFP like protein. PCT/GB96/00481describes the expression of GFP in plants. PCT/US95/01425 describesmodified GFP protein expressed in transformed organisms to detectmutagenesis. U.S. Pat. Nos. 5,401,629 and 5,436,128 describe assays andcompositions for detecting and evaluating the intracellular transductionof an extracellular signal. Recombinant cells that express cell surfacereceptors and contain reporter gene constructs that includetranscriptional regulatory elements that are responsive to the activityof cell surface receptors are used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the components of the cell-based scanningsystem.

FIG. 2 shows a schematic of the microscope subassembly

FIG. 3 shows the camera subassembly

FIG. 4 illustrates cell scanning system process

FIG. 5 illustrates a user interface showing major functions to guide theuser

FIG. 6 illustrates data presentation on screen

FIG. 7 flow chart of processing step for the cell-based scanning system

FIG. 8A-J illustrates the strategy of the Nuclear Translocation Assay

FIG. 9 is example data from a known inhibitor of translocation

FIG. 10 is example data from a known stimulator of translocation

SUMMARY OF THE INVENTION

The invention relates to a computer controlled optical-mechanical systemfor rapidly determining the distribution, environment, or activity offluorescently labeled reporter molecules in cells for the purpose ofscreening large numbers of compounds for those that specifically affectparticular biological functions. The invention involves:

-   -   providing cells containing fluorescent reporter molecules in an        array of locations,    -   treating the cells in the array of locations with one or more        reagents,    -   imaging numerous cells in each location with a fluorescence        microscope,    -   converting the optical information into digital data,    -   utilizing the digital data to determine the distribution,        environment or activity of the fluorescently labeled reporter        molecules in the cells and the distribution of the cells, and    -   interpreting that information in terms of a positive, negative        or null effect of the compound being tested on the biological        function

The array of locations may be a microtiter plate or a microchip which isa microplate having cells in an array of locations. The inventionincludes an apparatus and a computerized method for acquiring data suchas a digital frame grabber, processing, displaying and storing the data.

DETAIL DESCRIPTION OF THE INVENTION

Screening large numbers of compounds for activity with respect to aparticular biological function requires preparing arrays of cells forparallel handling of cells and reagents. Standard 96 well microtiterplates which are 86 mm by 129 mm, with 6 mm diameter wells on a 9 mmpitch, are used for compatibility with current automated loading androbotic handling systems. The microplate is typically 20 mm by 30 mm,with cell locations that are 100-200 microns in dimension on a pitch ofabout 500 microns. Methods for making microplate are described in U.S.Ser. No. 60/018,696 filed on May 30, 1996, assigned to the sameassignee. This application is incorporated herein by reference in itsentirety. Microplates may consist of coplanar layers of materials towhich cells adhere patterned with materials to which cells will notadhere, or etched 3-dimensional surfaces of similarly patteredmaterials. For the purpose of the following discussion, the terms ‘well’and ‘microwell’ refer to a location in an array of any construction towhich cells adhere and within which the cells are imaged. Microplatesalso include fluid delivery channels in the spaces between the wells.The smaller format of a microplate increases the overall efficiency ofthe system by minimizing the quantities of the reagents, storage andhandling during preparation and the overall movement required for thescanning operation. In addition, the whole area of the microplate can beimaged more efficiently, allowing a second mode of operation for themicroplate reader as described later in this document.

Those skilled in this art will recognize a wide variety of ways to makeand deliver fluorescent reporter molecules to cells. For example,fluorescently labeled biomolecules such as proteins, phospholipids andDNA hybridizing probes, as well as fluorescent reagents specificallysynthesized with particular chemical properties of binding orassociation have been used as fluorescent reporter molecules.Fluorescently labeled antibodies are particularly useful reportermolecules due to their high degree of specificity for attaching to asingle molecular target in a mixture of molecules as complex as a cell,tissue or extract of either.

Fluorescently labeled reporter molecules are useful for determining thelocation, amount and chemical environment of the reporter. For example,whether the reporter is in a lipophilic membrane environment or in amore aqueous environment can be determined. The pH environment of thereporter can be determined. It can be determined whether a reporterhaving a chelating group is bound to an ion, such as Ca++, or not.

Those skilled in the art will recognize a wide variety of ways tomeasure fluorescence. For example, some fluorescent reporter moleculesexhibit a charge in excitation or emission spectra, some exhibitresonance energy transfer where one fluorescent reporter loosesfluorescence, while a second gains in fluorescence, some exhibit a loss(quenching) or appearance of fluorescence, while some report rotationalmovements.

As mentioned earlier, see Description of the Prior Art above, a cell canbe genetically engineered to express reporter molecules such as GFPcoupled to a protein of interest. Using the method and systems of thepresent invention permits the determination of the site and extent ofactivity of the protein in the cells.

FIG. 1 is a schematic diagram of the system for measuring thedistribution, environment, or activity of fluorescent reporter moleculesin cells. An inverted fluorescent microscope 1 is a Zeiss Axiovertinverted fluorescence microscope which uses standard objectives withmagnification of 1-100× to the camera, and a white light source (e.g.100 W mercury-arc lamp or 75 W xenon lamp) with power supply 2. There isan XY stage 3 to move the plate 4 in the XY direction over themicroscope objective. A Z-axis focus drive 5 moves the objective in theZ direction for focusing. A joystick 6 provides for manual movement ofthe stage in the XYZ direction. A high resolution digital camera 7acquires images from each well or location on the plate. 8 is a camerapower supply. 9 is an automation controller and 10 is a centralprocessing unit. The PC 11 provides a display 12 and has associatedsoftware. The printer 13 provides for printing of a hard copy record. 14are the microscope oculars.

FIG. 2 is a schematic of the microscope assembly 1 showing in moredetail the XY stage 3, Z-axis focus drive 5, joystick 6, light source 2,automation controller 9, oculars 14. 15 and 16 are cables to thecomputer and microscope, respectively. In addition, FIG. 2 shows a 96well microtiter plate 17 which is moved on the XY stage 3 in the XYdirection. Light from the light source 2 passes through the PCcontrolled shutter 18 to a motorized filter wheel 19 with excitationfilters 20. The light passes into filter cube 25 which has a dichroicmirror 26 and an emission filter 27. Excitation light reflects off thedichroic mirror to the wells in the microtiter plate 17 and fluorescentlight 28 passes through the dichroic mirror 26 and the emission filter27 and to the digital camera 7.

FIG. 3 shows a schematic drawing of the camera assembly. The digitalcamera 7, which contains an automatic shutter for exposure control,receives fluorescent light 28 from the microscope assembly. A digitalcable 30 transports digital signals to the computer. 31 is the camerapower supply.

FIG. 4 illustrates an alternative embodiment of the invention in whichcells are in microwells 40 on a microplate 41. Typically the microplateis 20 mm by 30 mm as compared to a standard 96 well microtiter platewhich is 86 mm by 129 mm. The microplate chamber 42 serves as amicrofluidic delivery system for the addition of compounds to cells. Themicroplate 41 in the microplate chamber 42 is placed in an XY microplatereader 43. Digital data is processed as described above. The small sizeof this microplate system increases throughput, minimizes reagent volumeand provides for the ability to control the distribution and placementof cells for fast and precise cell-based analysis. This information canbe displayed on a PC screen 11 and made part of a bioinformatics database 44. This data base is an important part of the present inventionbecause it not only permits storage and retrieval of data obtainedthrough the methods of this invention, but also permits acquisition andstorage of external data relating to cells. FIG. 5 is a PC display whichillustrates the operation of the software.

In addition to the advantages cited earlier, the higher density array ofcells on a microplate allows the microplate reader to image the wholemicroplate at a low resolution of a few microns per pixel for highthroughput and image particular locations on the microplate at a higherresolution of less than 0.5 microns per pixel for higher precision.These two resolution modes improve the overall throughput of the system.

Referring to FIG. 7, at the start of an automated scan, the operatorenters information 100 that describes the sample, specifies the filtersettings and fluorescent channels to match the biological labels beingused and the information sought and then adjusts the camera settings tomatch the sample brightness. For flexibility to handle a range ofsamples, the software next allows selection of various parametersettings used to identify nuclei, cytoplasm, different fluorescentreagents, cell selection settings and number of cells to be analyzed perwell. These parameters are stored in the system's database for easyretrieval for each automated run. The system's interactive cellidentification mode simplifies the selection of morphological parameterlimits such as the range of size, shape, and intensity of cells to beanalyzed. The user specifies which wells of the plate the system willscan and how many fields or how many cells to analyze in each well.Depending on the setup mode selected by the user at step 101, the systemeither automatically pre-focuses the region of the plate to be scannedusing an autofocus procedure to “find focus” of the plate 102 or theuser interactively pre-focuses 103 the scanning region by selectingthree “tag” points which defines the rectangular area to be scanned bythe system. A least-squares fit “focal plane model” is then calculatedby the system from these tag points to estimate the focus of each wellduring a an automated scan. The focus of each well is estimated byinterpolating from the focal plane model during a scan.

During an automated scan, the software dynamically displays the statusof scan in progress such as the number of cells that have been analyzed,the current well that is being analyzed, and images of each independentwavelength as they are acquired, and the result of the screen for eachwell as it is acquired. The plate 4 is scanned in a serpentine style asthe software automatically moves the motorized microscope XY stage 3from well to well and field to field within each well of a 96-wellplate. Those skilled in the programming art will recognize how to adaptsoftware for scanning of other microplate formats such as 24, 48, and384 well plates. The scan pattern of the entire plate as well as thescan pattern of fields within each well are programmed. The systemadjusts sample focus with an autofocus procedure 104 through the Z axisfocus drive 5, controls filter selection via a motorized filter wheel 19and acquires and analyzes images of up to four different colors(“channels” or “wavelengths”).

The autofocus procedure is called at a user selected frequency,typically for the first field in each well and then once every 4 to 5fields within each well. The autofocus procedure calculates the startingZ-axis point by interpolating from the pre-calculated plane focal model.Starting a programmable distance above or below this set point, theprocedure moves the mechanical Z-axis through a number of differentpositions, acquires an image at each, and finds the maximum of acalculated focus score that estimates the contrast of each image. The Zposition of the image with the maximum focus score determines the bestfocus for a particular field. Those skilled in the art will recognizethis as a variant of automatic focusing algorithms as described in theprior art in Harms et al. in Cytometry 5 (1984), p. 236-243, Groen etal. in Cytometry 6 (1985), p. 81-91, and Firestone et al. in Cytometry12 (1991), p. 195-206.

For acquisition of images, the camera's 7 exposure time is separatelyadjusted for each dye to ensure a high-quality image from each channel.Software procedures can be called, at the user's option, to correct forregistration shifts between wavelengths by accounting for linear (X andY) shifts between wavelengths before making any further measurements.The electronic shutter 18 is controlled so that sample photo-bleachingis kept to a minimum. Background shading and uneven illumination canalso be corrected by the software using algorithms known in the priorart.

In one channel, images are acquired of a primary marker 105 (typicallycell nuclei counterstained with DAPI or PI fluorescent dyes) which aresegmented (“identified”) using an adaptive thresholding procedure. Theadaptive thresholding procedure 106 is used to dynamically select thethreshold of an image for separating cells from the background. Thestaining of cells with fluorescent dyes can vary to an unknown degreeacross cells in a microtiter plate sample as well as within images offield of cells within each well of a microtiter plate. This variationcan occur due to sample preparation and/or as a result of the nature ofcell biology. A global threshold is calculated for the complete image toseparate the cells from background. The global adaptive techniques usedin the system are variants of those described in prior art in Kittler etal. in Computer Vision, Graphics, and Image Processing 30 (1985), p.125-147, Ridler et al. in IEEE Trans. Systems, Man, and Cybernetics(1978), p. 630-632. These global adaptive thresholding techniquesaccount for field to field variation. Another adaptive thresholdingalgorithm in the system utilizes local region thresholding in contrastto global image thresholding. Image analysis of local regions in theimage leads to better overall segmentation since staining of cell nuclei(as well as other labeled components) can vary across an image. Theglobal/local processing model increases throughout and improves cellsegmentation. Using this global/local procedure, a reduced resolutionimage (reduced in size by a factor of 2 to 4) is first globallysegmented (using adaptive thresholding) to find regions of interest inthe image. These regions then serve as guides to more fully analyze thesame regions at full resolution. A more localized threshold is thencalculated (using again adaptive thresholding) for each region ofinterest.

The output of the segmentation procedure is a binary mask wherein theobjects are white and the background is black. This binary image, alsocalled a mask in the prior art, is used to determine if the fieldcontains objects 107. The mask is labeled with a blob labeling algorithmwhereby each object (or blob) has a unique number assigned to it.Morphological features, such as area and shape, of the blobs are used todifferentiate blobs likely to be cells from those that are consideredartifacts. The user pre-sets the morphological selection criteria byeither typing in known cells morphological features or by using theinteractive training utility. If objects of interest are found in thefield, images are acquired for all other active channels 108, otherwisethe stage is advanced to the next field 109 in the current well. Eachobject of interest is located in the image for further analysis 110. Thesoftware determines if the object meets the criteria for a valid cellnucleus 111 by measuring its morphological features (size and shape).For each valid cell, the XYZ stage location is recorded, a small imageof the cell is stored, and features are measured 112. The cell scanningsystem can perform multiple tests on cellular samples by applying anumber of analysis methods simultaneously to measure features atmultiple wavelengths including:

-   -   1. the total fluorescent intensity within the cell nucleus for        colors 1-4    -   2. the area of the cell nucleus for color 1 (the primary marker)    -   3. the shape of the cell nucleus for color 1 is described by        three shape features:        -   a) perimeter squared area        -   b) box area ratio        -   c) height width ratio    -   4. the average fluorescent intensity within the cell nucleus for        colors 1-4 (i.e. #1 divided by #2)    -   5. the total fluorescent intensity of a ring outside the nucleus        (see FIG. 8) that represents fluorescence of the cell's        cytoplasm (cytoplasmic mask) for colors 2-4    -   6. the area of the cytoplasmic mask    -   7. the average fluorescent intensity of the cytoplasmic mask for        colors 2-4 (i.e. #5 divided by #6)    -   8. the ratio of the average fluorescent intensity of the        cytoplasmic mask to average fluorescent intensity within the        cell nucleus for colors 2-4 (i.e. #7 divided by #4)    -   9. the difference of the average fluorescent intensity of the        cytoplasmic mask and the average fluorescent intensity within        the cell nucleus for colors 2-4 (i.e. #7 minus #4)    -   10. the number of fluorescent domains (also call spots, dots, or        grains) within the cell nucleus for colors 2-4

Features 1 through 4 are commonly used in a variety of image analysisapplications and are well known in prior art. Features 5-9 have beendeveloped specifically to provide measurements of a cell's fluorescentmolecules within the local cytoplasmic region of the cell and thetranslocation (i.e. movement) of fluorescent molecules from thecytoplasm to the nucleus. These screen specific features are used foranalyzing cells in microplates for the inhibition of nucleartranslocation. Inhibition of nuclear translocation of transcriptionfactors provides a novel approach to screening intact cells. Anautomated screen of an inhibitor of NF-κB translocation has beensuccessfully performed. A specific algorithm measures the amount ofNF-κB probe in the nuclear region (feature 4) versus the localcytoplasmic region (feature 7) of each cell. Quantification of thedifference between these two sub-cellular compartments provides ameasure of cytoplasm-nuclear translocation (feature 9).

Feature 10 is used for counting of DNA or RNA probes within the nuclearregion in colors 2-4. For example, DNA probes are commercially availablefor identifying the centromeres of specific chromosomes. Cells arethree-dimensional in nature and when examined at a high magnificationunder a microscope one probe may be in-focus while another may becompletely out-of-focus. The cell screening system has a procedure fordetecting three-dimensional probes in nuclei by acquiring images frommultiple focal planes. The software moves the Z-axis motor drive 5 insmall steps where the step distance is user selected to account for awide range of different nuclear diameters. At each of the focal steps,an image is acquired. The maximum gray-level intensity from each pixelin each image is found and stored in a resulting maximum projectionimage. The maximum projection image is then used to count the probes.The above algorithm work well in counting probes that are not stackeddirectly above or below another one. To account for probes stacked ontop of each other in the Z-direction, users can select an option toimprove the counting of probes by analyzing probes in each of the focalplanes acquired. In this mode, the scanning system performs the maximumplane projection algorithm as discussed above, detects probe regions ofinterest in this image, then further analyzes these regions in all thefocal plane images.

After measuring cell features 112, the systems checks if there are anyunprocessed objects in the current field 113. If there are anyunprocessed objects, it locates the next object 110 and checks if itmeets the criteria for a valid cell nucleus 111 and measures itsfeatures. After it the system has processed all the objects in thecurrent field, it checks if it is done with the current plate 114. Ifthe system is not done with the current plate, it check if it needs tofind more cells in the current well 115. If it needs to find more cellsin the current well it advances the XYZ stage to the next field withinthe current well 109 or it advances the stage to the next well 116 ofthe plate.

After a scan of a plate is complete, images and data can be reviewedwith the system's image review, data review, and summary reviewfacilities. All images, data, and settings from a scan are archived inthe system's database for later review. Users can review the imagesalone of every cell analyzed by the system with an interactive imagereview procedure 117. The user can review data on a cell-by-cell basisusing a combination of interactive graphs, a data spreadsheet offeatures measured, and images of all the fluorescent channels of a cellof interest with the interactive cell-by-cell data review procedure 118.Graphical plotting capabilities are provided in which data can beanalyzed via interactive graphs such as histograms and scatter plots.Users can review summary data that are accumulated and summarized forall cells within each well of a plate with an interactive well-by-welldata review procedure 119. Hard copies of graphs and images can beprinted on a wide range of standard printers. All images and data arestored in a the system's database for archival and retrieval or forinterface with a network laboratory management information system. Datacan also be exported to other third-party statistical packages totabulate results and generate other reports.

As a final phase of a complete scan, reports can be generated on one ormore statistics of features measured. Users can generate a graphicalreport of data summarized on a well-by-well basis for the scanned regionof the plate using an interactive report generation procedure 120. Thisreport includes a summary of the statistics by well in tabular andgraphical format and identification information on the sample. Thereport window allows the operator to enter comments about the scan forlater retrieval. Multiple reports can be generated on many statisticsand be printed with the touch of one button. Reports can be previewedfor placement and data before being printed.

EXAMPLE SCREEN 1 Automated Screen for Compounds that Induce or InhibitNuclear Translocation of a DNA Transcription Factor

Regulation of transcription of some genes involves activation of atranscription factor in the cytoplasm, resulting in that factor beingtransported into the nucleus where it can initiate transcription of aparticular gene or genes. This change in transcription factordistribution is the basis of a screen for the cell-based screeningsystem to detect compounds which inhibit or induce transcription of aparticular gene or group of genes. A general description of the screenis given followed by a specific example.

The distribution of the transcription factor is determined by labelingthe nuclei with a DNA specific fluorophore like Hoechst 33423 and thetranscription factor with a specific fluorescent antibody. Afterautofocusing on the Hoechst labeled nuclei, an image of the nuclei isacquired in the cell-based screening system at 20× magnification andused to create a mask by one of several optional thresholding methods.The morphological descriptors of the regions defined by the mask arecompared with the user defined parameters and valid nuclear masks areidentified and used with the following algorithm to extracttranscription factor distributions. Each valid nuclear mask is eroded todefine a slightly smaller nuclear region. The original nuclear mask isthen dilated in two steps to define a ring shaped region around thenucleus, which represents a cytoplasmic region. The average antibodyfluorescence in each of these two regions is determined, and thedifference between these averages is defined as the NucCyt Difference.Two examples of determining nuclear translocation are discussed belowand illustrated in FIG. 8A-J. FIG. 8A illustrates an unstimulated cellwith its nucleus 200 labeled with a blue fluorophore and a transcriptionfactor in the cytoplasm 201 labeled with a green fluorophore. FIG. 8Billustrates the nuclear mask 202 derived by the cell-based screeningsystem. FIG. 8C illustrates the cytoplasm 203 of the unstimulated cellimaged at a green wavelength. FIG. 8D illustrates the nuclear mask 202is eroded (reduced) once to define a nuclear sampling region 204 withminimal cytoplasmic distribution. The nucleus boundary 202 is dilated(expanded) several times to form a ring that is 2-3 pixels wide that isused to define the cytoplasmic sampling region 205 for the same cell.FIG. 8E further illustrates a side view which shows the nuclear samplingregion 204 and the cytoplasmic sampling region 205. Using these twosampling regions, data on nuclear translocation can be automaticallyanalyzed by the cell-based screening system on a cell by cell basis.FIG. 8F-J illustrates the strategy for determining nuclear translocationin a stimulated cell. FIG. 8F illustrates a stimulated cell with itsnucleus 206 labeled with a blue fluorophore and a transcription factorin the cytoplasm 207 labeled with a green fluorophore. The nuclear mask208 in FIG. 8G is derived by the cell based screening system. FIG. 8Hillustrates the cytoplasm 209 of a stimulated cell imaged at a greenwavelength. FIG. 8I illustrates the nuclear sampling region 211 andcytoplasmic sampling region 212 of the stimulated cell. FIG. 8J furtherillustrates a side view which shows the nuclear sampling region 211 andthe cytoplasmic sampling region 212.

A specific application of this method has been used to validate thismethod as a screen. A human chondrocyte cell line was plated in 96 wellmicrotiter plates. Some rows of wells were titrated with IL-1α, a knowninducer of the nuclear transcription factor NF-κB. The cells were thenfixed and stained by standard methods with a fluorescein labeledantibody to NF-κB, and Hoechst 33423. The cell-based screening systemwas used to acquire and analyze images from this plate and the NucCytDifference was found to be strongly correlated with the amount of IL-1αadded to the wells as illustrated in FIG. 9. In a second experiment, anantagonist to the receptor for IL-1, IL-1RA was titrated in the presenceof IL-1α, progressively inhibiting the translocation induced by IL-1α.The NucCyt Difference was found to strongly correlate with thisinhibition of translocation, as well as illustrated in FIG. 10.

Additional experiments have shown that the NucCyt Difference givesconsistent results over a wide range of cell densities and reagentconcentrations, and can therefore be routinely used to screen compoundlibraries for specific nuclear translocation activity. Furthermore, thesame method can be used with antibodies to other transcription factors,or GFP-transcription factor chimeras, in living and fixed cells, toscreen for effects on the regulation of transcription of this and othergenes.

FIG. 6 is a representative display on a PC screen of data which wasobtained in accordance with Example 1. Graph 1 300 plots the differencebetween the average antibody fluorescence in the nuclear sampling regionand cytoplasmic sampling region, NucCyt Difference verses Well #. Graph2 301 plots the average fluorescence of the antibody in the nuclearsampling region, NP1 average, versus the Well #. Graph 3 302 plots theaverage antibody fluorescence in the cytoplasmic sampling region, LIP1average, versus Well #. The software permits displaying data from eachcell. For example, FIG. 5 shows a screen display 406, the nuclear image401, and the fluorescent antibody image 402 for cell #14.

NucCyt Difference referred to in graph 1 303 of FIG. 6 is the differencebetween the average cytoplasmic probe (fluorescent reporter molecule)intensity and the average nuclear probe (fluorescent reporter molecule)intensity. The invention provides a computer means for converting thedigital signal from the camera into this parameter and for plotting theparameter verses the well number.

NP1 average referred to in graph 2 304 of FIG. 6 is the average ofcyloplasmic probe (fluorescent reporter molecule) intensity within thenuclear sampling region. The invention provides a computer means forconverting the digital signal from the camera into this parameter andfor plotting the parameter verses the well number.

L1P1 average referred to in graph 3 305 of FIG. 6 is the average probe(fluorescent reporter molecule) intensity within the cytoplasmicsampling region. The invention provides a computer means for convertingthe digital signal from the camera into this parameter and for plottingthe parameter verses the well number.

EXAMPLE SCREEN 2 Automated Screen for Compounds that Induce or InhibitHypertrophy in Cardiac Myocytes

Hypertrophy in cardiac myocytes has been associated with a cascade ofalterations in gene expression and can be characterized in cell cultureby an alteration in cell size, that is clearly visible in adherent cellsgrowing on a coverslip. Preliminary experiments indicate that a screencan be implemented using the following strategy. Myocytes cultured in 96well plates, can be treated with various compounds and then fixed andlabeled with a fluorescent antibody to a cell surface marker and a DNAlabel like Hoechst. After focusing on the Hoechst labeled nuclei, twoimages are acquired, one of the Hoechst labeled nuclei and one of thefluorescent antibody. The nuclei are identified by thresholding tocreate a mask and then comparing the morphological descriptors of themask with a set of user defined descriptor values. Local regionscontaining cells are defined around the nuclei. The limits of the cellsin those regions are then defined by a local dynamic threshold operationon the same region in the fluorescent antibody image. A sequence oferosions and dilations is used to separate slightly touching cells and asecond set of morphological descriptors is used to identify singlecells. The area of the individual cells is tabulated in order to definethe distribution of cell sizes for comparison with size data from normaland hypertrophic cells. In addition, a second fluorescent antibody to aparticular cellular protein, such as one of the major muscle proteinsactin or myosin can included. Images of this antibody can be acquiredand stored with the above images, for later review, to identifyanomalies in the distribution of these proteins in hypertrophic cells,or algorithms can be developed to automatically analyze thedistributions of the labeled proteins in these images.

Additional Screens

Those skilled in the art will recognize a wide variety of distinctscreens that can be developed. There is a large and growing list ofknown biochemical and molecular processes in cells that involvetranslocations or reorganizations of specific components within cells.The signaling pathway from the cell surface to target sites within thecell involves the translocation of plasma membrane-associated proteinsto the cytoplasm. For example, it is known that one of the src family ofprotein tyrosine kinases, pp60c-src, translocates from the plasmamembrane to the cytoplasm upon stimulation of fibroblasts withplatelet-derived growth factor (PDGF). In contrast, some cytoplasmiccomponents translocate from the cytoplasm to the plasma membrane uponstimulation of cells. For example, it is known that the GTP-bindingproteins of the Rho family are maintained as cytoplasmic complexes withRhoGDI in resting cells, but are released and translocate to plasmamembrane during cell activation. In addition, specific organelles, suchas components of the cytoskeleton, nuclear envelope, chromatin, golgiapparatus, mitochondria, and endosomes are reorganized in response tospecific stimuli. Finally, the targets for screening can themselves beconverted into fluorescence-based reagents that report molecular changesincluding ligand-binding and post-translocational modifications.

REFERENCES

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1. A non-transitory machine readable storage medium comprising a programcontaining a set of instructions when executed cause a cell screeningsystem to execute procedures for detecting translocation of a cellularcomponent of interest between a first cellular compartment and a secondcellular compartment on and/or within cells, wherein the procedurescomprise: a) providing an array of locations that contain multiplecells, wherein the cells contain a plurality of fluorescent reportermolecules, wherein the plurality of fluorescent reporter moleculescomprises optically distinguishable fluorescent reporter molecules thatreport on: i) individual cells; ii) the first cellular compartment; iii)the second cellular compartment; and iv) a cellular component ofinterest; wherein the fluorescent reporter molecules that report on thecellular component of interest are optically distinguishable from thefluorescent reporter molecules that report on individual cells, thefirst cellular compartment, and the second cellular compartment; b)automatically scanning multiple cells in each of the locationscontaining cells to obtain fluorescent signals from the plurality offluorescent reporter molecules; c) automatically identifying cells fromthe fluorescent signals; d) automatically defining a first cellularcompartment mask and a second cellular compartment mask in the cellsfrom the fluorescent signals; e) automatically determining an intensityof the fluorescent signals from the fluorescent reporter molecules thatreport on the cellular component of interest; and f) automaticallydetermining one or both of the following: i) a ratio of the intensity ofthe fluorescent signals from the fluorescent reporter molecules thatreport on the cellular component of interest in the first cellularcompartment mask and the second cellular compartment mask; and ii) adifference of the intensity of the fluorescent signals from thefluorescent reporter molecules that report on the cellular component ofinterest in the first cellular compartment mask and the second cellularcompartment mask; wherein the ratio of the intensity of the fluorescentsignals from the fluorescent reporter molecules that report on thecellular component of interest in the first cellular compartment maskand the second cellular compartment mask and/or the difference of theintensity of the fluorescent signals from the fluorescent reportermolecules that report on the cellular component of interest in the firstcellular compartment mask and the second cellular compartment maskprovides a measure of the translocation of the cellular component ofinterest between the first cellular compartment and the second cellularcompartment on and/or within the cells.
 2. The machine readable storagemedium of claim 1, wherein the ratio of the intensity of the fluorescentsignals from the fluorescent reporter molecules that report on thecellular component of interest in the first cellular compartment maskand the second cellular compartment mask and/or the difference of theintensity of the fluorescent signals from the fluorescent reportermolecules that report on the cellular component of interest in the firstcellular compartment mask and the second cellular compartment maskprovides a measure of test compound-induced translocation of thecellular component of interest between the different cellularcompartments on and/or within the cells.
 3. The machine readable storagemedium of claim 1, wherein the first cellular compartment and the secondcellular compartment consist of a cell nucleus and a cell cytoplasm, andwherein the translocation consists of a translocation between the cellcytoplasm and the cell nucleus.
 4. The machine readable storage mediumof claim 1 wherein the first cellular compartment and the secondcellular compartment consist of a cell cytoplasm and a cell membrane,and wherein the translocation consists of a translocation between thecell cytoplasm and the cell membrane.
 5. The machine readable storagemedium of claim 1, wherein the plurality of fluorescent reportermolecules comprise fluorescent reporter molecules expressed by thecells.
 6. The machine readable storage medium of claim 1, wherein theplurality of fluorescent reporter molecules are expressed by the cells.7. The machine readable storage medium of claim 1, wherein the pluralityof fluorescent reporter molecules comprise fluorescent reportermolecules added to the cells.
 8. The machine readable storage medium ofclaim 1, wherein the plurality of fluorescent reporter molecules areadded to the cells.
 9. The machine readable storage medium of claim 1wherein the plurality of fluorescent reporter molecules compriseantibodies.
 10. The machine readable storage medium of claim 1 whereinthe plurality of fluorescent reporter molecules comprise compounds thatreport on DNA.
 11. The machine readable storage medium of claim 1wherein the cellular component of interest comprises a protein.
 12. Themachine readable storage medium of claim 11 wherein the proteincomprises a transcription factor.
 13. The machine readable storagemedium of claim 1 wherein steps (b) through (f) are performed atmultiple time points.